环境中细微颗粒物对人支气管上皮细胞转分化的影响

PM2.5 from traffic-related ambient air and wood smoke induces epithelial-mesenchymal transition in human bronchial epithelial cells

目的 初步观察交通主干道旁大气细微颗粒物（TAPM2.5）和使用木材烹饪厨房内细微颗粒物（WSPM2.5）对人支气管上皮细胞（HBEC）上皮-间充质细胞转分化（EMT）的影响.方法 分别采集交通主干道旁和使用木材烹饪的厨房内大气中的细颗粒物,通过DMSO萃取获得TAPM2.5-DMSO和WSPM2.5-DMSO.用不同浓度TAPM2.5-DMSO和WSPM2.5-DMSO刺激HBEC 24 h后,应用细胞增殖-毒性检测试剂盒（CCK-8）检测细胞活力;通过细胞免疫荧光和蛋白免疫印迹法,检测TAPM2.5-DMSO（20 mg/L）、WSPM2.5-DMSO（10 mg/L）刺激HBEC 14 d后,上皮细胞标志物E-钙黏素蛋白（E-cadherin）、细胞角蛋白（Cytokeratin）和间充质细胞标志物α-平滑肌肌动蛋白（α-SMA）、波形蛋白和Ⅰ型胶原蛋白（COL-Ⅰ）的表达情况.结果 TAPM2.5-DMSO（浓度为1、2、10 mg/L）和WSPM2.5-DMSO（浓度为1、5、10 mg/L）对细胞活力产生影响,增强细胞的活力[分别为（118.4±13.7）％、（118.2±8.0）％、（123.0±19.6）％和（112.4±4.1）％、（120±5.4）％、（117.8±7.0）％,F值分别为71.4和49.09,均P＜0.05],在TAPM2.5-DMSO（20 mg/L）和WSPM2.5-DMSO（15和20mg/L）刺激组细胞活力处于平衡状态[（100.7±12.1）％和（106.8±10.0）％、（93.8±7.9）％,均P＞0.05].TAPM2.5-DMSO浓度达到100、200 mg/L,WSPM2.5-DMSO浓度达到30、40 mg/L后,对细胞活力逐渐呈现抑制作用[分别为（53.4±15.3）％、（9.4±1.7）％和（60.9±9.5）％、（46.2±3.6）％,均P＜0.01].与空白对照组相比,TAPM2.5-DMSO和WSPM2.5-DMSO刺激组细胞形态发生改变,细胞免疫荧光提示上皮细胞标志物E-cadherin和Cytokeratin表达减少,间质细胞标志物α-SMA和波形蛋白表达上调;蛋白免疫印迹提示Cytokeratin表达水平在两组均明显下调[（0.063±0.109）和（0.039±0.313）,P值分别为0.033和0.030],E-cadherin表达呈下调趋势,但差异无统计学意义[（0.862±0.096）和（0.817±0.212）,P值分别为0.228和0.117];PM2.5刺激组COL-Ⅰ表达均上调[（2.549±1.037）和（3.658±1.207）,P值分别为0.034和0.001],α-SMA仅在WSPM2.5-DMSO刺激组表达明显上调（8±5,P=0.049）.结论 交通主干道旁大气PM2.5和木材烟雾PM2.5均可引起人支气管上皮细胞发生上皮-间充质细胞转分化改变,木材烟雾PM2.5的作用似乎更为显著.

Objective To observe if arterial traffic ambient PM2.5 （TAPM2.5） and wood smoke PM2.5 （WSPM2.5） exposure can induce epithelial-mesenchymal transition （EMT） in human bronchial cells （HBEC）.Methods PM2.5 was collected from an arterial traffic road and a typical southern kitchen,and then the collections were extracted by DMSO.The viability of HBEC was measured by Cell Counting Kit （CCK-8） after culture with PM2.5-DMSO extracts for 24 hours.The expressions of EMT markers,including E-cadherin,cytokeratin,α-smooth muscle actin （α-SMA）,vimentin and collagen type Ⅰ （COL-Ⅰ） in HBEC were assayed by cell immunofluorescence and Western blot analysis after exposed to two different sources of PM2.5-DMSO extracts for 14 days.Results The cell viability of HBEC increased at low concentrations （1,2,10 μg/ml and 1,5,10 μg/ml,corresponding to [（118.4 ± 13.7）%,（118.2 ± 8.0）%,（123.0±19.6）% and （112.4±4.1）%,（120±5.4）%,（117.8±7.0）%,respectively,allP〈 0.05],and then declined at high levels [20,100,200 iμ g/ml and 15,20,30,40 μg/ml,corresponding to（100.7±12.1）%,（53.4±15.3）%,（9.4±1.7）% and （106.8±10.0）%,（93.8±7.9）%,（60.9± 9.5）%,（46.2 ± 3.6）%,respectively,P values were 0.923,0.000,0.000 and 0.231,0.278,0.000,0.000,respectively] in both TAPM2.5-DMSO and WSPM2.5-DMSO incubation.After exposure for 14 days,the cells lost their typical cobblestone-like shape which implied that EMT might occur.The same treatment caused decreased positive signals of E-cadherin and cytokeratin in a small proportion of the cells.The decreasedexpressions of cytokeratin were verified by Western blot （TAPM2.5 and WSPM2.5 were 0.063 ± 0.109 and0.039 ± 0.313,P values were 0.033 and 0.030,respectively）,while α-SMA was only significantlyupregulated in the WSPM2.5-DMSO group （7.853 ± 4.784,P =0.049）.The expressions of E-cadherindecreased in both groups but not statistically significant in Western blot （0.862 ±0.096 and 0.817 ±0.212,P values were 0.228 and 0.117,respectively）.Another marker of EMT,COL-Ⅰ,markedly increased in both PM2.5 treatment groups （2.549 ± 1.037 and 3.658 ± 1.207,P values were 0.034 and 0.001）.Conclusions Both PM2.5 from arterial traffic ambient air and wood smoke could induce EMT in human bronchial epithelial cells,while WSPM2.5 appeared to have a more significant influence on EMT in HBEC.